Plant Cell - Transient Gene Expression in lntact and ...592 The Plant Cell the small subunit of...

The Plant Cell, Vol. 2, 591-602, July 1990 O 1990 American Society of Plant Physiologists

Transient Gene Expression in lntact and Organized Rice Tissues

Rudy A. Dekeyser, Bart Claes, Riet M.U. De Rycke, Marianne E. Habets, Marc C. Van Montagu,' and Allan B. Caplan Laboratorium voor Genetica, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, 8-9000 Gent, Belgium

Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a 8-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light- harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.

INTRODUCTION

Despite recent reports of the production of transgenic maize (Rhodes et al., 1988) and rice (Zhang and Wu, 1988; Shimamoto et al., 1989), transformation of monocotyledonous plants is neither as routine nor as reproducible as that of dicotyledonous plants (Potrykus, 1989). As a consequence, analysis of regulatory sequences or of the effects of modifying biochemical pathways through introduction of new genes into Gramineae has been very limited.

Transient gene expression (TGE) assays provide an alternative system to analyze genetically modified plant genes. This approach has used electroporation (Fromm et al., 1985) or polyethylene glycol treatments (Shillito et al., 1985) to introduce genes transiently into plant protoplasts so that their expression can be evaluated. TGE assays offer two advantages over analysis of stable transform- ants. The major advantage is undoubtedly that gene activity can be measured within hours or days after DNA introduction. By comparison, it takes severa1 weeks or months before stably transformed lines are available for detailed study. Furthermore, because the vast majority of transferred DNA remains extrachromosomal during the time course of a TGE assay (Werr and Lorz, 1986), the analysis is not confounded by influences exerted by chro- mosomal sequences adjacent to the sites of integrated

' To whom correspondence should be addressed.

genes. In transgenic plants these so-called position effects can influence both the leve1 and the specificity of newly introduced genes (Jones et al., 1985; Poulsen et al., 1986).

Whereas inducible gene expression has been analyzed with TGE assays in protoplasts (e.g., Callis et al., 1988; Huttly and Baulcombe, 1989), the question remains whether regulated expression in cells without walls and dissociated from their original organs and tissues is always comparable with the expression in the corresponding intact tissues. Protoplast isolation treatments disrupt the developmental control systems dependent on cell/cell contact or on the position of a cell in an organized structure. Moreover, once protoplasts begin to resynthesize cell walls and divide in culture, they converge to the same phenotype regardless of their origin. This leads to loss of the differentiated state and quite probably of the control processes unique to those tissues. All of these problems are compounded in monocotyledons because the isolation of high-quality protoplasts from differentiated tissue has proven difficult. As a consequence, the standard material for TGE assays in monocotyledons is derived from cell suspension cultures, precluding examination of differential gene expression in different organs.

Severa1 studies demonstrate the limitations of TGE assays in protoplasts. For example, mesophyll protoplasts isolated from illuminated tobacco leaves do not synthesize

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592 The Plant Cell

the small subunit of ribulose-1,5-bisphosphate carboxylase(Fleck et al., 1979; Vernet et al., 1982). Similarly, leafprotoplasts from barley leaves do not synthesize abscisicacid in response to osmotic stress, although the intactleaves do (Loveys and Robinson, 1987). In addition, vary-ing terminator sequences of a chimeric gene varied geneexpression as much as 60-fold in transgenic tobaccoleaves but almost not at all in electroporated tobacco leafprotoplasts (Ingelbrecht et al., 1989).

In the hope of circumventing the limitations of TGEassays with protoplasts and to be able to study regulatedgene expression in monocotyledons, we modified existingelectroporation conditions in such a way that it becamepossible to deliver DMA into organized and intact planttissue. This novel protocol not only retained all the advan-tages of a TGE assay, but allowed us to investigate, forthe first time, the expression of both a light-regulated anda tissue-specific chimeric gene in monocotyledons. Theapproach was developed for rice, but could easily beadapted for maize, wheat, and barley.

RESULTS

Optimization of DMA Introduction into Intact Cells

Based on previous experiments (Dekeyser et al., 1989),we used a construct (p2'-nptll) with the promoter fromthe mannopine synthase 2' gene of the AgrobacteriumTR-DNA (Velten et al., 1984) fused to the neomycin phos-photransferase II (nptll) gene (Beck et al., 1982) to assesswhether transient gene expression could be detected inintact and organized tissue.

Leaf base explants from 7-day-old, etiolated rice seed-lings, as shown in Figure 1, were chosen to test thepermeability of plant cells because (1) the explants containseveral types of differentiated cells making it possible tostudy tissue-specific expression if the DMA were intro-duced uniformly, (2) the tissue contains meristematic cellscapable of developing into embryogenic callus tissue (Wer-nickeetal., 1981; Abdullahetal., 1986) so that proliferativecapacity after treatment could be monitored, (3) the tissueis soft, indicating a less rigid cell wall, and (4) the explantsare easily obtained.

In initial experiments, 25 leaf bases were electroporatedusing conditions similar to those appropriate for proto-plasts (Dekeyser et al., 1989). After 2 days of culture, theelectroporated leaf bases showed low levels of neomycinphosphotransferase II (NPTII) activity (data not shown).Leaf bases electroporated with a promoterless nptll con-struct never showed any NPTII activity. To improve theefficiency of DNA introduction, we analyzed (1) factorsinfluencing the DNA/explant interaction, (2) physical pa-rameters related to the electroporation technique, and (3)chemical parameters that might protect the DNA or en-hance the viability of the treated tissue.

SLE

PR

Figure 1. Gross Anatomy of a 7-Day-Old Etiolated Rice Seedling.

LBE, position of leaf base explant; SLE, position of sheath/laminaexplant; PR, primary root; SR, secondary root.

Figure 2A illustrates that preincubating the isolated leafbases in electroporation (EPR) medium before adding theplasmid DNA was essential for efficient introduction ofDNA. When the preincubation time was prolonged from15 min to 180 min, a 25-fold increase in NPTII activity wasobtained. This result is at least partially related to theleakage of nucleases out of the leaf bases, a phenomenonalso observed in excised barley leaves (Galston et al.,1979). This was demonstrated by refreshing the electro-poration buffer after 15 min, 30 min, 60 min, 120 min, and180 min, incubating each fraction with 2 ^g of supercoiledplasmid DNA for 1 hr, and assessing the integrity of theplasmid DNA by agarose gel electrophoresis. Whereas theplasmid DNA incubated with the 180-min fraction remainedintact, there was progressively more degradation of thesamples in medium removed at the earlier time points (datanot shown). Plasmid DNA degradation was even morepronounced when the experiment was done with rootexplants.

As shown in Figure 2B, another fourfold increase inNPTII activity was obtained by expanding the coincubationtime of the plasmid DNA with the leaf bases from 15 minto 60 min.

The number or size of the pores in electroporated cells

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Electroporation in lntact Tissue 593

A

preincubotion time (hours)

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O 100 300 500 700 900

copacilorsize ( { f F )

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. . O 1 2 3

co-incubation time (hours)

D - &O 1 0 0 , s 100

i z

- - $ 0

o 5 0 100 150 a o

5 0 100 150

concentrat ion NoCl (mM)

F

eieclr icd field (Volt/cm) concentrolion of Spermidine (mM)

Figure 2. Relationship between the Electroporation Parameters and the Leve1 of NPTll Activity in Electroporated Leaf Bases.

Each point represents the amount of radioactive phosphorylated kanamycin detected with that condition relative to the maximum obtained within that experiment and averaged over at least two experiments. If not noted otherwise, the leaf bases were electroporated with the standard protocol. (A) Effect of incubation of leaf bases in electroporation buffer before electroporation. (6) lnfluence of the time leaf bases are incubated with the plasmid DNA. (C) Effect of the capacitor size and the number of pulses (W, one pulse; O, three pulses; A, five pulses). (D) Effect of the concentration of NaCl in the electroporation buffer during the electrical discharge (A, with a 900-pF capacitor; O, with a 200-pF capacitor). (E) Effect of electrical field strength delivered from different-size capacitors (A, 200 pF; W, 500 pF; O, 900 pF). (F) lnfluence of the addition of spermidine to all solutions.

is correlated with the duration and the amplitude of the electrical pulse provided. Figure 2C illustrates that with a constant pulse amplitude (375 V/cm) and EPR medium containing 150 mM NaCI, maximal NPTll activity was reached with a 900-pF capacitor. This corresponds to a pulse lasting approximately 300 msec. Pulse times higher than 300 msec, which were obtained by retaining the 900- pF capacitor and decreasing the concentration of NaCl in the EPR medium, decreased the level of NPTll activity (Figure 2D). Figure 2D also shows that using a 200-pF capacitor and EPR medium without NaCl yielded levels of

NPTll activity comparable with those obtained with a 900- pF capacitor and 150 mM NaCl in the EPR buffer. The pulse time for both conditions was very similar. lncreasing the number of pulses only elevated the level of NPTll activity when the pulse duration was short (Figure 2C). At higher pulse durations, this benefit was lost, quite probably because of the increased damage to the cells.

The effect of amplitude on transient expression is sum- marized in Figure 2E. If the duration of the pulses was extended (900 pF, 150 mM NaCI, and to a lesser extent 500 pF, 150 mM NaCI), the NPTll activity peaked at an electrical field of 375 V/cm. Under optimal DNA introduction conditions (900 wF, 150 mM NaCI, and 375 V/cm), 33% of the electroporated leaf bases remained capable of initiating callus tissue on LS2.5 medium. This is approximately 50% of the callus initiation observed with unelec- troporated leaf base fragments.

Experiments employing DNA concentrations as Iow as 5 pg/mL produced readily detectable amounts of NPTll activity. Enzyme levels increased linearly as DNA concentrations were raised to 100 pg/mL (data not shown). Both super- coiled and linearized plasmid DNA provided similar levels of NPTll activity. Plasmids used in this paper ranged from 3.5 kb to 15 kb, but the correlation between plasmid size and cell penetration has not been systematically studied.

A time course of gene expression after electroporation showed that NPTll activity after DNA introduction rose to a maximum at 4 days and could be easily detected as early as 1 day and as late as 8 days after electroporation (data not shown).

In an attempt to further improve the efficiency of DNA transfer, we experimented with chemicals that might protect DNA against degradation by nucleases or enhance the viability of the treated tissue. Figure 2F shows that enriching incubation and electroporation solutions with 0.2 mM spermidine, which delays lysis of oat protoplasts and inhibits the activity of nucleases (Galston et al., 1979), improved the NPTll signal threefold. Two compounds that have been used to enhance transformation efficiencies in animal cells, protamine (Wienhues et al., 1987) and chloroquine diphosphate (Luthman and Magnusson, 1983), did not improve the gene transfer efficiency in electroporated leaf bases. All requirements for optimal expression based on these studies are chronologically outlined in Methods.

Transient Expression in Leaf Bases from Other Cereals

To prove that the delivery of DNA to intact cells has broad applications, we subjected leaf bases from other monocotyledonous crop plants to the same electroporation procedure. Figure 3 shows that high enzyme activity was obtained in maize and wheat using either the p2’-npt// or a similar construct driven by the cauliflower mosaic virus 35s promoter. Higher expression might be obtained in

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594 The Plant Cell

Rice Maize Wheat Barley

"5<}

<f *s<?* > <' ?<?' <V

Figure 3. General Applicability of Electroporation for IntroducingDNA into Gramineae.Shown is an autoradiogram of an NPTII assay on leaf bases fromvarious cereals electroporated according to the standard proto-col with equimolar amounts of either p2'-nptll or p35S-nptllconstructs.

barley or any of the other species by modifying the elec-troporation conditions, but the precise optimization pro-cedure has not been pursued in these studies.

Transient Expression in Other Leaf Tissues

To determine whether the electroporation procedure wasmore generally applicable, we tested four types of ex-plants: leaf bases (LBD) and sheath/lamina tissue from firstand second leaves (SLD) from etiolated seedlings (Figure1), and leaf bases (LBL) and sheath/lamina tissue (SLL)from etiolated seedlings transferred to the light for 8 hr.

Using the optimized conditions for leaf bases from etiol-ated seedlings, we electroporated the p2'-nptll and p35S-nptll constructs into the different explants, quantified theNPTII activity according to Reiss et al. (1984), and nor-malized the results from every independent experiment tothe NPTII activity produced in LBD explants electroporatedwith the p2'-nptll gene. As illustrated in Table 1, thevariation in relative NPTII activity in three independentexperiments is low for LBD, LBL, and SLD explants. Thevariability determined in extracts from SLL explants is atleast partially due to the low absolute NPTII levels, whichmake accurate measurements difficult. Based on theseexperiments, the average relative level of NPTII activity inLBD, LBL, SLD, and SLL tissue amounts to 100%, 93%,18%, and 4%, respectively, when electroporated with thep2'-nptll construct, and to 98%, 95%, 17%, and 5%,respectively, when electroporated with the p35S-nptllgene. These results are shown in Figure 4. A third promoterderived from a Nicotiana plumbaginifolia extensin genefused with the same nptll reporter sequence (M. De Loose,unpublished results) directs 20 times less activity in LBD

explants (Table 1). These results show that the 2' and35S promoters are similarly expressed in rice leaf tissuealthough it should be noted that in protoplasts the 2'promoter was consistently found to be 3 times strongerthan the 35S promoter (Dekeyser et al., 1989).

Light-Regulated Expression

To analyze whether the electroporated leaf bases weresufficiently unperturbed to allow photoregulated geneexpression, a construct consisting of the promoter and thetransit peptide sequence from the light-inducible peaIhcpABdO gene fused to the nptll gene (Van den Broecket al., 1988) was electroporated into the four differentexplants (Figure 4). Whereas no enzyme activity was de-tected in etiolated explants, NPTII activity in the green SLLtissue reached levels averaging 3 times to 5 times higherthan those obtained with the p2'-nptll and p35S-nptllgenes (Table 1). This demonstrates that the expressionfrom the pea promoter sequence is light-regulated in leaftissue from rice. Light, however, is not the only factorgoverning the expression in this tissue: we were unable todetect NPTII activity in leaf bases from light-grown seed-lings. To determine whether this tissue-dependent differ-ence mirrors the expression of the endogenous light-harvesting chlorophyll proteins (LHCPs), all tissues wereexamined cytologically.

Using the electron microscope, LBL tissues were foundto possess amyloplasts but no chloroplasts, as shown in

Table 1. Variability of NPTII Activity in Intact Leaf TissueElectroporated with Chimeric Genes

p2'-nptll

p35S-nptll

pext-nptll

plhcp-nptll

LBD

100100100105929756

DD"UDUD

LBL

9710082

1029389NDa

NDUDUDUD

SLD

181521171718NDNDUDUDUD

SLL

254734

NDND8

1027

Experiment12312312123

Four different leaf explants from 7-day-old rice seedlings havebeen electroporated with four chimeric genes in three independentexperiments. The NPTII activity present in the electroporatedtissues was analyzed (Reiss et al., 1984) and normalized to theNPTII activity in leaf bases from dark-grown seedlings (LBD)electroporated with the p2' -nptll construct. See Figure 4 fordescription of other explants." ND, not determined.b UD, not detected.

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Electroporation in Intact Tissue 595

Q

p2'-nptll

p35S-nptII

plhcp-nptll

Figure 4. Developmental Control over Transient Gene Expression.

Autoradiogram of an NPTII assay performed on leaf bases (LB)or mixed leaf sheath and lamina tissue (SL) isolated from dark-grown (D) or light-grown (L) seedlings and electroporated withequimolar amounts of photo-insensitive (p2'-nptll and p35S-nptll)or photo-regulated (plhcp-nptll) reporter genes.

Figure 5F. In contrast, the SLL mesophyll tissue containedchloroplasts with well-developed thylakoid membranesand grana (Figures 5B and 5D). The cells from the etiolatedLBD and SLD explants contained amyloplasts and etio-plasts, respectively (Figures 5A, 5C, and 5E). The devel-opment of mature photosynthetic organelles was found tocoincide with the synthesis of the endogenous rice LHCP.With antibodies raised against the pea LHCP, which havebeen shown to cross-react with Arabidopsis, tobacco, andalfalfa LHCPs (G. Engler, personal communication), wecould readily detect LHCP associated with the thylakoidmembranes of chloroplasts in mesophyll cells from thesheath/lamina tissue of light-grown seedlings (Figures 5Band 5D). No cross-reacting protein could be detected inparenchyma cells of any of the other explants.

We conclude that the success or failure to detect NPTIIactivity from the plhcp-nptll constructs correlates with theexpression of the endogenous rice Ihcp gene(s). In addi-tion, photoregulation of both the endogenous and foreigngenes depends in part on the developmental stage of theplastids, as was previously noticed in tobacco (Simpsonetal., 1986).

The mobility of the LHCP-NPTII protein in the enzymaticassay was greater than expected for the fusion proteinand, in fact, migrated where the protein would be if the

Ihcp transit peptide had been removed. This might indicatethat the NPTII protein is targeted to the chloroplasts, butthis has not been rigorously demonstrated here. However,the ribulose-1,5-bisphosphate carboxylase small subunittransit peptide from pea targets the NPTII protein to chlo-roplasts of barley mesophyll protoplasts (Teeri et al., 1989),so the ability of monocotyledonous cells to utilize dicotchloroplasts transit sequences may be a generalphenomenon.

Generalized Permeability of Electroporated Cells toDMA Transfer

None of the 150 rice leaf bases electroporated in theabsence of DMA and incubated with 5-bromo-4-chloro-3-indolyl-/3-D-glucuronic acid (X-gluc) showed /3-glucuroni-dase (GUS) activity in any tissue, as shown, for example,in Figure 6A. On this basis, leaf bases were electroporatedwith the f3-glucuronidase gene (Jefferson et al., 1987)under control of either the cauliflower mosaic virus (CaMV)35S promoter (p35S-uidA) or the 2' promoter (p2'-uidA),stained with X-gluc 4 days after treatment, and analyzedto determine which cells were susceptible to DNA intro-duction. At the macroscopic level, these electroporatedleaf bases displayed, like electroporated protoplasts (Gallieet al., 1989), variation in expression of the 0-glucuronidasegene. Five percent to 20% of the leaf bases electroporatedwith either of the two chimeric constructs produced GUSat detectable levels as judged by the presence of blue-stained cells. Of the positively reacting leaf bases, 5% to20% stained uniformly blue, the others only showed sec-tors of blue-stained cells. We believe that the differentialexpression levels in the leaf bases might be at leastpartially related to the position of the tissue in the electro-poration chamber and to the fact that each fragmentpossesses a nonuniform resistance. Consequently, differ-ent leaf bases may receive slightly different electricalpulses.

Histological examination of several leaf bases demon-strated that both complete and partially stained leaf basesshowed a similar cellular expression pattern. In leaf baseselectroporated with the p2'-uidA construct, high 0-glucu-ronidase activity was detected in inner and outer epider-mis, parenchyma, and vascular bundle cells (Figure 6B).This illustrates that the 2' promoter is similarly expressedin different cell types from the rice leaf base and that allcells, ranging from approximately 10 ^m to 100 urn indiameter, can be permeabilized by the electrical discharge.It also proves that one electrical pulse can introduce DNAsimultaneously into more than six layers of cells. The p35S-uidA gene was similarly expressed (Figures 6C and 6D),further indicating that the DNA is randomly introduced, notpreferentially delivered, to a specific cell type. Becausethere is also no clear correlation between the cut surfaces

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596 The Plant Cell

Figure 5. Degree of Photomaturation in 7-Day-Old Rice Explants.

(A) and (C) Chloroplast development in mesophyll cells in leaf sheath and leaf lamina from dark-grown rice seedlings.(B) and (D) Chloroplast development in mesophyll cells in leaf sheath and leaf lamina from light-grown rice seedlings.(E) Structure of parenchyma cells in dark-grown leaf bases.(F) Structure of parenchyma cells in light-grown leaf bases.All sections have been cross-reacted with antibodies against the pea LHCP and treated subsequently with 15-nm, protein-A gold todemonstrate that the LHC protein has been synthesized in light-grown sheath and lamina tissues. The abbreviations used are: ap,amyloplast; Chl, chloroplast, cw, cell wall; cyt, cytoplasm; ep, etioplast; er, endoplasmic reticulum; m, matrix, mt, mitochondria; N, nucleus;Nu, nucleolus; th, thylakoid membrane; Va, vacuole. The black bar in Figures A to F is 1 nm.

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and the sites of staining, the plasmid DNA is not entering the explants exclusively where cell walls have been phys- ically damaged.

Tissue-Specific Expression

In Arabidopsis and tobacco, a construct (psam-uidA) containing the promoter from the Arabidopsis S-adenosylmethionine synthetase (sam) gene fused to the bacterial uidA coding region is preferentially expressed in vascular tissue (Peleman et al., 1989a, 1989b). Therefore, this reporter system was chosen to assess whether tissue-specific gene expression can be demonstrated after electroporation. RNA gel blot analysis, using the coding region of a rice sam gene as a probe, verified that the endogenous sam gene family was expressed in 4-day-old to 8-day-old seedlings (data not shown).

At the macroscopic level, the staining pattern generated by psam-uidA-electroporated leaf bases was different from the pattern in p2 ’4dA- and p35S-uidA-electroporated explants. From 250 treated leaf bases, seven randomly se- lected blue-stained leaf bases were sectioned and analyzed histologically. All of them showed a similar expression pattern, as illustrated in Figures 6E to 61. The vast majority of the GUS activity was restricted to cells surrounding the vascular bundles. Occasionally, we observed some randomly stained epidermis cells, but none of the leaf bases showed the nearly uniform staining seen with 35s or 2’ promoters.

DISCUSSION

Electroporation is a very efficient technique to introduce DNA into many types of protoplasts. Previous reports have indicated that intact plant cells can be permeabilized by electroporation. For instance, sugar beet suspension cultures electroporated with a chloramphenicol acetyltrans- ferase (cat) gene showed CAT activities 2 times above background, but higher levels were only obtained after treatment with pectolytic enzymes (Lindsey and Jones, 1987). This type of enzymatic treatment could be as disruptive for promoter regulation as complete cell wall removal. This approach contrasts with the one reported in this paper in which DNA is delivered efficiently into un- treated plant cells using electroporation. It is not clear whether all plants have susceptible tissues, but our approach has succeeded for rice, maize, wheat, and barley. Under optimized conditions, the NPTll activity directed by the p2’-nptll and p35S-nptll reporter genes amounts to more than 1 O0 times the background activity.

Plasmid DNA might be entering cells in any of severa1 ways. A priori (1) the DNA might enter only damaged cells, or (2) the electrical discharge may be able to disrupt both

cell walls and cell membranes creating pores in both barriers, or (3) the DNA might penetrate passively through pores of the cell wall during the incubation period with leaf material so that the electrical shock is needed only to permeabilize the membrane. Whereas the first hypothesis is unlikely because we could demonstrate that the expression of the introduced reporter genes is not restricted to wound sites, we cannot discriminate between the second and the third hypotheses at this time.

Three modifications of the rice protoplast electroporation conditions were necessary to obtain optimal nucleic acid transfer into intact leaf cells. First, excised tissue had to be preincubated for 3 hr in electroporation buffer before transfer to fresh medium containing the plasmid DNA to remove nucleases excreted by damaged cells. Second, the coincubation of the plasmid DNA with the plant material had to be prolonged. This probably reflects the slow dif- fusion of DNA into the intercellular spaces and/or the slow penetration of DNA through the cell wall pores. Third, efficient DNA introduction required a pulse duration up to 300 msec, which is 5 times to 10 times higher than the pulses used to electroporate protoplasts.

We initially monitored the expression of two commonly used chimeric genes, the p2’-nptll and the p35S-nptll. In rice explants, the highest expression of either promoter was obtained in leaf bases (LB) from both dark- (D) and light-grown (L) seedlings. In the etiolated sheath and lamina tissue from the first and second foliar leaves (SLD), the expression of both constructs was reduced fivefold, and in green sheath and lamina tissue (SLL), the NPTll activity reached only 5% of the activity in leaf bases. Because light can stimulate changes in the cell wall (Kim et al., 1989), it seems likely that the explant-dependent differences are at least partially due to differences in the efficiency of entry of DNA. On the other hand, because tissue-specific expression is maintained in these explants, we cannot exclude the possjbility that some of the difference is caused by explant-specific cellular signals.

In contrast to the transcript 2’ and CaMV 35s promoters, the light-inducible pea lhcpAB80 promoter is highly expressed in light-exposed, chloroplast-containing SLL tissue from rice and is not expressed at detectable levels elsewhere. The expression seems to correlate with the expression of the endogenous rice lhcp genes that were monitored immunologically. Whereas it has been reported that a monocotyledonous lhcp promoter is photoregulated in transgenic tobacco (Lamppa et al., 1985), we provide evidence that a dicotyledonous promoter is appropriately regulated in a monocotyledonous tissue. This further sup- ports the idea that the components of the phototransduc- tion pathway and the promoter sequences necessary for light-regulated expression have been highly conserved among distantly related species. Nevertheless, differences exist that need further study because a wheat ribulose- 1,5-bisphosphate carboxylase small subunit promoter was not expressed in transgenic tobacco plants (Keith and

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598 The Plant Cell

oep ,V

B

pa

iep

vx^mmmm

Figure 6. Potential for Tissue-Specific Gene Expression in Electroporated Leaf Bases.

(A) Leaf base explants electroporated without DNA. The abbreviations used are: iep, inner epidermis; oep, outer epidermis; pa, parenchyma;vb, vascular bundle. Magnification X100.

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Chua, 1986), and neither the pea nor the Arabidopsis small subunit promoters were able to provide detectable NPTll activity in electroporated green leaf tissue (data not shown). Our failure to detect pssu-npW expression might be due to evolutionary differences between the different plants or may indicate that further modifications need to be made to the leaf tissue electroporation system.

Indirectly, the light-inducible expression of the lhcpAB80 promoter implies that plasmid DNA could be electroporated into rice mesophyll cells. More precise information about the susceptibility of the cells to electroporation- mediated gene transfer has been obtained by histological analysis of leaf base explants electroporated with p-glucuronidase reporter genes.

The p35S-uidA gene was expressed in cells of the inner and outer epidermis, parenchyma, and vascular bundles of rice. A similar expression pattern of the CaMV 35s promoter has been observed in transgenic tobacco leaf tissue (Benfey et al., 1989), suggesting that the regulatory sequences of this promoter are conserved sufficiently to permit uniform expression in both monocotyledon and dicotyledon leaves. The uniformity of this expression and that shown by p2’-uidA also demonstrates that plasmid DNA can be delivered into leaf explants without significant bias. Consequently, reproducible nonuniform cellular expression patterns obtained after chimeric genes have been electroporated into leaf bases most likely reflect differential gene regulation rather than differential DNA introduction. For example, in all sections investigated, the psam-uidA construct was preferentially expressed in cells within and surrounding the vascular strands. This agrees with the expression pattern of the psam-uidA construct in transgenic Arabidopsis and tobacco leaves (Peleman et al., 1989a, 1989b), indicating that the regulatory mecha- nism for this type of expression is most probably not perturbed by our TGE assay.

The wide range of cells that can express DNA after electroporation and the observation that one electrical pulse can introduce nucleic acids simultaneously into more than six cell layers lead us to conclude that the potential of electroporation-mediated gene transfer might match the potential of the biolistic method (Klein et al., 1987). The particle gun protocol has the advantage that it uses less DNA and that it has been more elaborately investigated (for a review, see Sanford, 1988); our method offers the advantage that it can be performed with a simple, com-

monly available electroporation apparatus, that it has a high penetration power into adjacent cell layers, and that there is no need to construct DNA-binding tungsten particles.

Based on the expression of the p2’-uidA and p35S-uidA constructs, it seems likely that plasmid DNA can enter the meristematic leaf base cells located between the vascular bundle and the lower epidermis (Morrish et al., 1987). The frequency of this event, the amount of DNA entering the cell, and the competence of these cells to integrate foreign DNA into their genome will determine the probability of recovering transgenic embryogenic callus and, consequently, transformed rice plants. The suitability of this method to obtain transformed callus tissue from electroporated leaf bases and to transfer DNA into other meristematic explants, such as immature embryos, is under investigation.

METHODS

Plant Material

Seeds from rice (Oryza sativa cv Taipei 309) kindly supplied by the lnternational Rice Research lnstitute (Manila, Philippines) were sterilized according to Thompson et al. (1 986). To germinate, seeds were transferred to Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 1% sucrose and 0.8% Difco agar, and grown in the dark at 25%. If greening of seedlings was necessary, 6-day-old plants were transferred to the light for 8 hr. Wheat (Triticum aestivum cv Fidel), barley (Hordeum vulgare cv Gerbel), and maize (Zea mays line 2418) seeds were sterilized for 30 min in 4.5% commercial bleach, rinsed five times in sterile water, and grown like rice seeds. Wheat and barley seeds were obtained from NV Clovis Matton (Adelgem, Belgium) and the maize seeds from line 241 8 produced by de Cooperative de Pau (Lescar, France).

Plasmids

CsCI-purified plasmid DNA was used in the electroporation experiments. The plasmid concentration was determined spectro- photometrically and verified by agarose gel electrophoresis. All chimeric constructs, with the exception of the p35S-uidA and p2’- uidA genes, which are terminated by the 3’ region of gene 7, are terminated by the 3’ region of the octopine synthase gene. The p2‘-nptll, p35S-nptl1, plhcp-nptll, and pext-nptll constructs are

Figure 6. (continued).

(8) Leaf base explants electroporated with plasmids encoding p2’-uidA. Magnification x l 00. (C) and (D) Leaf base explants electroporated with plasmids encoding p35S-uidA. Magnification xl O0 [(C)] and x250 [(O)]. (E), (F), (G), (H), and (I) Leaf base explants electroporated with plasmids encoding psam-uidA. Magnification x l00 [(E) and (F)] and x60 [ (G), (H), and (1)l. After 4 days leaf bases were stained with X-gluc and sectioned (see Methods). (A) through (F) Bright-field micrographs show transverse sections through isolated leaves. (G) through (I) Bright-field micrographs show transverse sections through the first three foliage leaves tightly rolled inside each other. Positive staining reactions show that the location of the blue precipitate depends on the construct used.

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600 The Plant Cell

carried by pLD1 (8.3 kb; Dekeyser et al., 1989), pGemOCS (3.5 kb; lngelbrecht et al., 1989), pGABTneo2 (8.2 kb; Van den Broeck et al., 1988), and pEN5 (6.4 kb; M. De Loose, unpublished results), respectively. lhe psam-uidA gene is carried by pCRSGUSl (6 kb; Peleman et al., 1989a). Thep2'-uidA construct resides on pGlucl (14.2 kb; Plant Genetic Systems NV) and thep35S-uidA gene on p260 (15 kb; P. Breyne, unpublished results). The promoter fragments used in these chimeric genes are similar to those used in the p2'-npf/l and p35S-nptll genes.

Electroporation of Leaf Bases

Twenty-five 7-day-old seedlings were used for each electroporation experiment. After removing the coleoptile, the leaf base region (4 mm) or the mixed sheath and lamina tissue (4 mm) (Figure 1) was isolated. lhe explants were transversely divided into 2-mm fragments and incubated for 3 hr in electroporation (EPR) buffer (10% glucose, 4 mM CaCI,, 10 mM Hepes, pH 7.2) with 0.2 mM spermidine. We then removed the EPR buffer, washed the explants twice with EPR buffer, and transferred them to a disposable spectrophotometer cuvette containing 0.2 mL of EPR buffer containing 0.2 mM spermidine. Twenty micrograms of pLDl DNA, or equimolar concentrations of the other plasmids, was incubated with the explants for 1 hr. Next, 11 pL of a 3 M NaCl stock was added and the cuvette was put on ice for 10 min. Parallel stainless steel electrodes, 2 mm thick and 6 mm apart, were inserted in such a way that the leaf bases were gathered between them. One pulse with an electrical field strength of 375 V/cm was discharged from a 900-pF capacitor. The homemade electroporation unit consisted of an ISCO power supplier connected with an array of capacitors arranged in a circuit, as described by Fromm et al. (1985). After 15 min on ice, explants were rinsed in KpR medium, pipetted to 5 mL of KpR medium (Thompson et al., 1986) with 2 mg/L 2,4-D, and incubated in the dark for 4 days. The explants from green seedlings were incubated in the light. The protocol was identical for the other species mentioned in this article, except that for maize only 17 explants were used. To test the frequency of callus induction of electroporated leaf bases, the concentration of glucose in the liquid KpR medium was reduced by 50% every day, and on the 4th day leaf bases were transferred to Linsmaier and Skoog (1965) medium with 3% sucrose, 0.4% agarose (type II, Sigma), and 2.5 mg/L 2,4-D (LS2.5).

Enzymatic Assays

NPTtl assays were performed according to Dekeyser et al. (1 989), except that leaf bases were crushed in 70 pL of 2 x ice-cold extraction buffer (1% p-mercaptoethanol, 50 mM Tris, pH 6.8, 0.13 mg/mL leupeptin). Quantification of the NPTll activity has been described by Reiss et al. (1 984).

Histochemical localization of GUS activity in electroporated leaf bases was performed essentially as described by Jefferson et al. (1 987). Four days after electroporation, leaf bases were immersed in 0.1 M sodium phosphate buffer (pH 7.2) containing 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-~-glucuronic acid (Research Organics Inc., Cleveland, OH). The reaction proceeded for 18 hr in the dark at 37OC. To stop the reaction, the tissue was rinsed three times in 0.1 M sodium phosphate buffer. Leaf bases were subsequently incubated in a 0.1 M sodium phosphate-buffered 1% glutaralde- hyde solution for 3 hr and rinsed once more in phosphate buffer.

Tissues were then embedded in 4% (w:v) agarose and sliced in 40-pm sections with a vibrocutter (Campden Instruments, Lon- don). Examination of the slices was performed with bright-field microscopy.

Electron Microscopy

Electron microscopy and immunochemistry were performed according to De Clercq et al. (1990). The pea LHCP antiserum was a generous gift of A. Cashmore and L. Szabo.

ACKNOWLEDGMENTS

We acknowledge Jean De Lafonteyne and Monika Sormann for the generous gift of seeds. The able technical assistance of Malvine Marichal is greatly appreciated. We thank Johan Botter- man (Plant Genetic Systems Inc.), Peter Breyne, Marc De Loose, and lvan lngelbrecht for the gift of plasmids before publication. The very skillful typing of the manuscript by Martine De Cock and the preparation of the figures by Vera Vermaercke, Karel Spruyt, and Stefaan Van Gijsegem are appreciated. We thank Roger De Vos for help in the construction of the capacitor unit. This work was supported by funds from the Rockefeller Foundation (RF 86058 No. 59), the Services of the Prime Minister (U.I.A.P. 120C0187), and the Commission of the European Communities [TS2-0053-B (GDF)]. R.D. is a research assistant of the National Fund for Scientific Research Belgium.

Received March 5, 1990; revised May 14, 1990.

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